β-Caryophyllene Protects Against Foodborne Ochratoxin A–Induced Pancreatic Toxicity: Implications for Food Safety and Environmental Health

β-Caryophyllene Protects Against Foodborne Ochratoxin A–Induced Pancreatic Toxicity: Implications for Food Safety and Environmental Health | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-Caryophyllene Protects Against Foodborne Ochratoxin A–Induced Pancreatic Toxicity: Implications for Food Safety and Environmental Health Peramaiyan Rajendran, Abdullah Al Zahrani, Ramya Sekar, Salaheldin Abdelraouf Abdelsalam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8885289/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The increasing prevalence of mycotoxin contamination due to climate change and environmental stressors represents a growing global health concern, necessitating effective preventive and therapeutic strategies. Ochratoxin A (OTA) is a pervasive foodborne mycotoxin linked to multisystem toxicity; nonetheless, its harmful effects on pancreatic tissue and the underlying molecular processes are inadequately defined. Increasing evidence indicates that oxidative stress and sterile inflammation produced by OTA are pivotal in pancreatic damage. β-Caryophyllene (BCP), a natural sesquiterpene found in consumable flora, demonstrates significant antioxidant and anti-inflammatory effects. The objective of this study was to examine the protective effect of BCP against OTA-induced pancreatic toxicity, focusing on the modulation of the Nrf2/HO-1 antioxidant pathway and the NF-κB-mediated activation of the NLRP3 inflammasome. Experimental animals were subjected to OTA to elicit pancreatic toxicity and simultaneously administered BCP at both low and high dosages. Pancreatic damage was assessed via serum biochemical markers and histological analysis. Parameters of oxidative stress and inflammatory mediators were measured in pancreatic tissue. Protein expression levels of Nrf2, HO-1, NF-κB, and NLRP3 signaling components were examined to clarify the molecular foundation of BCP-induced protection. OTA exposure caused significant pancreatic damage, indicated by increased oxidative stress, activation of NF-κB and NLRP3 inflammasome signaling, and notable histoarchitectural disruption. BCP therapy markedly reduced OTA-induced pancreatic damage in a dose-dependent fashion. BCP augmented intrinsic antioxidant defenses through the activation of the Nrf2/HO-1 pathway while simultaneously inhibiting NF-κB activation and subsequent NLRP3 inflammasome signaling. BCP significantly alleviates OTA-induced pancreatic damage by reestablishing redox equilibrium and suppressing inflammatory signaling pathways. These findings offer new understanding of OTA-induced pancreatic damage and recognize BCP as a potential dietary strategy for mitigating mycotoxin-related pancreatic impairment, with possible implications for health hazards connected with environmental and food safety. Mycotoxin Ochratoxin A β-Caryophyllene Pancreas NF-κB/NLRP3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The contamination of food and feed by mycotoxins represents a continual global hazard to human and animal health, especially in areas characterized by warm weather and insufficient storage conditions. Among these pollutants, ochratoxin A (OTA) is one of the most widespread and harmful secondary metabolites produced by Aspergillus and Penicillium species. OTA commonly contaminates cereals, coffee, wine, dried fruits, and animal feed, leading to persistent low-dose exposure via the food chain (Li et al. 2021 ; Peraica et al. 2010 ). While OTA is primarily known for its nephrotoxic, hepatotoxic, and carcinogenic characteristics, growing evidence suggests that its toxicological effects also impact extra-renal organs, such as the immune system, reproductive tissues, and pancreas (Mor et al. 2017 ; Zhu et al. 2026 ; Chen and Wu 2017 ). The pancreas is especially vulnerable to xenobiotic-induced damage because of its elevated metabolic requirements, abundant mitochondrial presence, and comparatively restricted antioxidant capability. Experimental studies have shown that OTA alters pancreatic redox equilibrium, causes lipid peroxidation, and inhibits insulin production, ultimately contributing to β-cell dysfunction and metabolic imbalance (Zanić et al. 1995; Ahmad et al. 2024 ; Fakhri et al. 2024 ). At the molecular level, exposure to OTA induces excessive formation of reactive oxygen species (ROS), mitochondrial dysfunction, and activation of inflammatory signaling pathways, ultimately resulting in pancreatic tissue damage and cellular apoptosis (Ou et al. 2023 ; Son et al. 2024 ). The exact processes connecting oxidative stress to the exacerbation of inflammation in OTA-induced pancreatic damage are not fully elucidated. The nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is a major regulator of cellular antioxidant defense, governing the transcription of numerous cytoprotective genes, including as heme oxygenase-1 (HO-1), superoxide dismutase, catalase, and glutathione-dependent enzymes Kuwar et al. 2025; AlZahrani et al.2024). In toxic or inflammatory conditions, the inhibition of Nrf2 signaling intensifies oxidative damage and enhances tissue susceptibility. In contrast, pharmacological or dietary stimulation of the Nrf2/HO-1 axis has demonstrated considerable protection against chemically induced organ toxicity, including pancreatic damage (Liu et al.2023; Luo et al. 2018 ). Oxidative stress is a crucial upstream initiator of inflammatory cascades, notably the nuclear factor-κB (NF-κB) pathway and the NOD-like receptor protein 3 (NLRP3) inflammasome, all of which play significant roles in pancreatic inflammation and dysfunction (Yang et el. 2020). Activation of NF-κB enhances the transcription of pro-inflammatory cytokines, whilst the assembly of the NLRP3 inflammasome facilitates caspase-1 activation and the maturation of interleukin-1β and interleukin-18, exacerbating tissue inflammation and damage. Increasing evidence indicates that dysregulated interaction between compromised Nrf2 antioxidant signaling and excessive activation of NF-κB/NLRP3 pathways is crucial in toxin-induced pancreatic injury and metabolic disorders (Papantoniou et al. 2024 ; Yang 2020). Therefore, agents capable of restoring redox balance while simultaneously suppressing inflammatory signaling are of considerable therapeutic interest. β-Caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene found in significant quantities in widely used spices, including black pepper, cloves, oregano, and cinnamon (Scandiffio et al. 2020 ; Sköld et al. 2005). BCP, a bioactive chemical obtained from food, has garnered significant interest due to its antioxidant, anti-inflammatory, and cytoprotective activities, as well as its excellent safety profile (Alonso et al. 2023 ; Scandiffio et al. 2020 ). Preclinical investigations have shown that BCP significantly reduces oxidative stress, inhibits NF-κB activation, suppresses NLRP3 inflammasome signaling, and maintains tissue architecture in diverse models of chemical- and toxin-induced organ injury (Scandiffio et al. 2020 ). Furthermore, recent research suggests that BCP can stimulate the Nrf2/HO-1 pathway, establishing it as a potential dual-action modulator of redox-inflammation imbalance. Although these promising attributes, the preventive function of β-caryophyllene against OTA-induced pancreatic damage has yet to be comprehensively investigated. The potential of BCP to mitigate OTA-induced pancreatic damage via the synchronized activation of Nrf2/HO-1 antioxidant mechanisms and the inhibition of NF-κB/NLRP3 inflammatory signaling is currently uncertain. It is crucial to address this gap to enhance our comprehension of dietary sesquiterpenes as prospective treatments for mycotoxin-related pancreatic and metabolic diseases. This study aimed to examine the protective benefits of β-caryophyllene against OTA-induced pancreatic toxicity in a mouse model. We posited that BCP alleviates pancreatic oxidative injury and inflammation via stimulating the Nrf2/HO-1 pathway while suppressing NF-κB/NLRP3 signaling. This study seeks to elucidate the mechanistic understanding of the redox-inflammatory modulation mediated by β-caryophyllene through comprehensive biochemical, molecular, and histopathological analyses, thereby endorsing its prospective use as a dietary or pharmacological intervention for OTA-induced pancreatic damage. 2. Materials and methods 2.1. Animals and ethical approval Adult male Swiss albino mice (8–10 weeks old; 25–30 g) by King Saud University’s animal house in Riyadh, Saudi Arabia. These pathogen-free, healthy mice acclimatized for one week in standard conditions (25 ± 2°C, 60–70% humidity, 12 h light/dark cycle) within polypropylene cages, with unlimited access to chow and water. All animal experimental procedures were conducted in strict accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and complied with the ARRIVE 2.0 guidelines. The study was conducted in accordance with internationally accepted ethical standards, including the U.K. Animals (Scientific Procedures) Act, 1986 and its associated guidelines, the EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The experimental protocol was reviewed and approved by the Animal Research Ethics Committee of King Faisal University, in accordance with the Declaration of King Faisal University, and approved by the Institutional Review Board (IRB) under approval number: ETHICS2877. 2.2. Chemicals and reagents Ochratoxin A (OTA; purity ≥ 99%) and β-caryophyllene (BCP; purity ≥ 98%) were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Antibodies for Western blotting and immunohistochemistry were sourced from Thermo Fisher Scientific (Waltham, MA, USA). Remaining chemicals and reagents were analytical grade from common commercial sources. OTA was dissolved in [0.5% DMSO in sterile saline] for intraperitoneal administration. BCP was suspended in [olive oil 0.5%] for oral gavage. All other reagents were of analytical grade. 2.3. Experimental design and treatment protocol Mice were randomly divided into five experimental groups (n = 6 per group). Group allocation was coded, and investigators involved in outcome assessment were blinded to treatment. Group I (Control): Vehicle 0.5% DMSO (oral gavage) Group II (OTA alone): OTA 3mg/kg/day (oral gavage) Group III (OTA + BCP low dose): OTA + BCP 25 mg/kg/day (oral gavage) Group IV (OTA + BCP-High dose): OTA + BCP high dose 50 mg/kg/day (oral gavage) Group V (BCP alone): BCP high dose 50 mg/kg/day (oral gavage) OTA was administered orally at [3 mg/kg] [once daily / x 3 day per week] for 6 weeks to induce pancreatic toxicity. BCP was administered by oral gavage daily throughout the OTA exposure period as a co-treatment regimen. The dosing strategy was selected based on published OTA toxicity models and the reported pharmacological safety of BCP (Norah et al. 2024; Hu et al. 2022 ). Animals were monitored daily for clinical signs of toxicity and mortality. Body weight was recorded weekly. 2.4. Sample collection At the end of the experimental period, mice were fasted overnight and euthanized under ketamine (80 mg/kg) anesthesia. Blood samples were collected by cardiac puncture, allowed to clot, and centrifuged at 3000 rpm for 10 min at 4°C to obtain serum. The pancreas was rapidly excised, rinsed in ice-cold PBS, blotted dry, and divided into portions for histopathology, biochemical analysis, and protein expression studies. Tissue samples were either fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at − 80°C. 2.5. Assessment of pancreatic injury markers Serum amylase and lipase levels were measured using commercial diagnostic kits purchased from Cayman Chemical Company (Ann Arbor, MI, USA), according to the manufacturer’s instructions. Fasting blood glucose levels were determined using a handheld glucometer, while serum insulin concentrations were quantified using a mouse insulin ELISA kit obtained from Cayman Chemical Company (Ann Arbor, MI, USA) to assess pancreatic endocrine function. 2.6. Evaluation of oxidative stress and antioxidant enzymes Pancreatic tissue homogenates (10% w/v) were prepared in ice-cold phosphate buffer (0.1 M, pH 7.4) and centrifuged at 10,000 ×g for 15 min at 4°C. The supernatant was used to determine MDA levels and the activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) using commercially available assay kits Cayman Chemical Company (Ann Arbor, MI, USA). Protein concentration was measured by the BCA method. 2.7. Histopathological examination Formalin-fixed pancreatic tissues were processed, embedded in paraffin, and sectioned at 4 µm thickness. Sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope Leica DM2500 LED. Pancreatic damage was evaluated by a blinded pathologist using a semi-quantitative scoring system assessing acinar cell degeneration, edema, inflammatory cell infiltration, and vacuolization. Representative photomicrographs were captured at 200× magnification and scale bar 50 µm. 2.8. Western blot analysis Total protein was extracted from pancreatic tissue using RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of protein (50 µg) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated overnight at 4°C with primary antibodies against Nrf2, HO-1, NF-κB p65, phospho-NF-κB p65, NLRP3, cleaved caspase-1, IL-1β, IL-18 and β-actin. After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence and quantified by densitometry using ImageJ software. 2.9. Statistical analysis Results are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant. Analyses were conducted using GraphPad Prism (version v8.4). 3. Result 3.1. BCP Attenuates OTA-Induced Hyperglycemia in Mice Figures 1 A and 1 B present the chemical structures of OTA and BCP, respectively. OTA exposure significantly increased fasting blood glucose levels compared with the control group ( p < 0.05) (Fig. 1 C), indicating metabolic dysregulation. BCP co-administration attenuated hyperglycemia in a dose-dependent manner, with the high-dose group showing a more pronounced reduction than the low-dose group. Serum insulin analysis (Fig. 1 D) revealed a significant decrease in OTA-treated animals ( p < 0.05), reflecting impaired β-cell function. BCP treatment significantly restored insulin levels in a dose-dependent fashion, with greater improvement observed at the higher dose. 3.2. BCP Mitigates OTA-Induced Pancreatic Injury via Suppression of Oxidative Stress and Inflammation Serum amylase (Fig. 2 A) and lipase (Fig. 2 B) levels were significantly elevated in the OTA-treated group compared with controls ( p < 0.05), confirming pancreatic exocrine injury. Co-administration of BCP markedly reduced both enzyme levels in a dose-dependent manner, with the high-dose group demonstrating near normalization. OTA exposure also significantly increased serum MDA levels (Fig. 2 C), indicating enhanced lipid peroxidation and oxidative stress ( p < 0.05). BCP treatment significantly attenuated MDA accumulation, suggesting restoration of redox homeostasis, potentially via activation of the Nrf2-mediated antioxidant defense system. Furthermore, serum TNF-α levels (Fig. 2 D) were markedly elevated in OTA-treated animals, reflecting systemic inflammatory activation. BCP supplementation significantly suppressed TNF-α in a dose-dependent manner, supporting inhibition of pro-inflammatory signaling, likely through modulation of the NF-κB pathway. Collectively, these findings indicate that BCP confers pancreatic protection against OTA toxicity by attenuating oxidative stress and inflammatory responses. 3.3. BCP Inhibits OTA-Induced Activation of NLRP3 and NF-κB Signaling Representative Western blot images of NLRP3 and NF-κB proteins are shown in Fig. 3 A. OTA exposure markedly upregulated the protein expression levels of both NLRP3 and NF-κB compared with the control group (p < 0.05), indicating activation of inflammasome formation and pro-inflammatory signaling in pancreatic tissue. Densitometric analysis of band intensities (Fig. 3 B), normalized to β-actin, confirmed a significant increase in NLRP3 and NF-κB protein expression in the OTA-treated group. Co-administration of BCP significantly downregulated both proteins in a dose-dependent manner. The high-dose BCP group exhibited a more pronounced suppression compared with the low-dose group (p < 0.05). These findings demonstrate that BCP attenuates OTA-induced pancreatic inflammation by inhibiting NF-κB activation and subsequent NLRP3 inflammasome signaling 3.4. BCP Restores Pancreatic Antioxidant Defense in OTA-Exposed Mice As shown in Fig. 4 A, OTA administration significantly increased pancreatic tissue MDA levels compared with the control group ( p < 0.05), indicating enhanced lipid peroxidation and oxidative damage. Co-treatment with BCP significantly reduced MDA levels in a dose-dependent manner, with the high-dose group exhibiting a more pronounced protective effect. Conversely, antioxidant enzyme activities—including SOD (Fig. 4 B), CAT (Fig. 4 C), and GPx (Fig. 4 D)—were significantly decreased in the OTA-treated group ( p < 0.05), reflecting impaired endogenous antioxidant defense. BCP supplementation markedly restored SOD, CAT, and GPx activities in a dose-dependent fashion, with greater normalization observed at the higher dose. These findings suggest that BCP mitigates OTA-induced oxidative injury by enhancing antioxidant capacity and re-establishing redox balance, likely through activation of the Nrf2-regulated antioxidant signaling pathway. 3.5. BCP on Histopathological Alterations in Pancreatic Tissue Group I (Control): The exocrine pancreas exhibited normal histological architecture, characterized by well-organized serous acini (black arrow) arranged in distinct lobules separated by intact fibrovascular septa (yellow arrow) (Fig. 5 ). Group II (OTA): Pancreatic tissue displayed severe disruption of normal architecture. Marked acinar cell degeneration with prominent cytoplasmic vacuolation (red arrow) was observed. Focal acinar atrophy with areas of necrosis were evident (blue arrow). Additionally, severe interstitial edema accompanied by mononuclear inflammatory cell infiltration and vascular congestion (green arrow) was present. Group III (OTA + BCP Low Dose): Moderate architectural disorganization was noted (Fig. 5 ). Acinar cell degeneration with cytoplasmic vacuoles persisted (red arrow). Large focal areas of hemorrhage with vascular congestion were observed (blue arrow), along with mild interstitial infiltration of mononuclear inflammatory cells (green arrow). Group IV (OTA + BCP High Dose): The pancreas showed mild architectural disorganization. Limited areas of acinar degeneration with cytoplasmic vacuolation were present (red arrow), with occasional focal necrosis (green arrow). Mild interstitial fibrosis with sparse mononuclear cell infiltration (blue arrow) was noted, indicating substantial histological improvement compared with the OTA group. Group V (BCP Alone): Pancreatic tissue exhibited normal histological features similar to the control group, with well-preserved serous acini (black arrow) arranged in lobules separated by intact fibrovascular septa (yellow arrow). 3.6. BCP Activates the Nrf2/HO-1/NQO1 Antioxidant Signaling Pathway Representative Western blot images of Nrf2, HO-1, and NQO1 protein expression are presented in Fig. 6 A. OTA exposure significantly reduced the expression levels of Nrf2 and its downstream antioxidant targets, HO-1 and NQO1, compared with the control group ( p < 0.05), indicating suppression of endogenous antioxidant defense mechanisms in pancreatic tissue. Densitometric analysis of band intensities (Fig. 6 B), normalized to β-actin, confirmed a significant downregulation of Nrf2, HO-1, and NQO1 in the OTA-treated group. Notably, BCP supplementation markedly restored the expression of these antioxidant proteins in a dose-dependent manner. The high-dose BCP group demonstrated a more pronounced upregulation compared with the low-dose group ( p < 0.05). These findings suggest that BCP confers protection against OTA-induced pancreatic oxidative injury by activating the Nrf2-mediated antioxidant signaling pathway 4. Discussion Mycotoxins are pervasive environmental contaminants produced by toxigenic fungi that proliferate under conditions of high humidity and temperature, making their occurrence particularly relevant in the context of climate change and global food insecurity (Marroquín et al 2014; Casu et al 2024 ; Shetty et al 2023 ). Environmental factors such as improper storage, agricultural practices, and rising temperatures significantly influence fungal growth and toxin production, leading to contamination of cereals, nuts, and animal feed. Chronic exposure to mycotoxins through the food chain poses substantial public health risks, including hepatotoxicity, nephrotoxicity, immunosuppression, and metabolic dysfunction (Awuchi et al 2022 ; Li et al 2025 ; Yilmaz et al 2025 ). Therefore, understanding the environmental determinants of mycotoxin contamination and developing effective mitigation strategies are critical for safeguarding food safety and reducing toxin-induced organ damage. The present study demonstrates that BCP effectively mitigates OTA-induced pancreatic toxicity through coordinated modulation of oxidative stress, inflammatory signaling, and inflammasome activation. OTA exposure resulted in marked hyperglycemia, reduced serum insulin levels, elevated pancreatic injury markers (amylase and lipase), enhanced lipid peroxidation, and suppression of endogenous antioxidant defenses. These biochemical alterations were accompanied by severe histopathological damage and activation of NF-κB/NLRP3 signaling, collectively indicating pancreatic dysfunction driven by oxidative and inflammatory mechanisms. OTA is a well-recognized mycotoxin that induces organ toxicity primarily via excessive ROS generation and redox imbalance. In the present study, elevated MDA levels together with decreased SOD, CAT, and GPx activities confirm oxidative stress as a central mechanism of OTA-induced pancreatic injury. The suppression of Nrf2 and its downstream targets HO-1 and NQO1 further supports impairment of the intrinsic antioxidant defense system. Nrf2 is a master regulator of cellular redox homeostasis, and its downregulation enhances susceptibility to oxidative damage and metabolic dysregulation (Goel et al 2025 ; Zhang et al 2025). Inflammation also played a critical role in OTA-mediated toxicity. Increased TNF-α levels and upregulated NF-κB expression indicate activation of pro-inflammatory transcriptional pathways. NF-κB is a pivotal regulator of cytokine production and inflammasome priming (Yoon et al 2023 ). Consistently, the elevated expression of NLRP3 suggests activation of the inflammasome complex, which amplifies inflammatory cascades and contributes to tissue degeneration (Wang et al 2025). Histopathological findings including acinar degeneration, necrosis, edema, and inflammatory infiltration corroborate the biochemical and molecular results. Importantly, BCP supplementation significantly attenuated OTA-induced pancreatic injury in a dose-dependent manner. BCP reduced hyperglycemia and restored serum insulin levels, suggesting preservation of endocrine function. It also normalized amylase and lipase levels, indicating protection of exocrine integrity. At the molecular level, BCP suppressed NF-κB activation and downregulated NLRP3 expression, thereby limiting inflammatory amplification. Concurrently, BCP restored Nrf2, HO-1, and NQO1 expression, enhancing antioxidant capacity and re-establishing redox balance. The reciprocal regulation between Nrf2 and NF-κB pathways likely underlies the protective mechanism of BCP. Activation of Nrf2 not only enhances antioxidant defense but also indirectly suppresses NF-κB mediated inflammatory signaling. Therefore, BCP appears to exert dual protective actions by both scavenging oxidative stress and inhibiting inflammatory signaling cascades. These findings are consistent with previous reports describing BCP as a natural sesquiterpene with potent antioxidant and anti-inflammatory properties. Collectively, the present data indicate that OTA-induced pancreatic toxicity is mediated by oxidative stress driven NF-κB activation and subsequent NLRP3 inflammasome signaling, whereas BCP confers significant protection through activation of the Nrf2/HO-1/NQO1 axis and suppression of inflammatory pathways. These findings highlight the therapeutic potential of BCP as a natural protective agent against mycotoxin-induced metabolic and pancreatic dysfunction. 5. Conclusion In conclusion, the present study demonstrates that ochratoxin A induces significant pancreatic injury characterized by hyperglycemia, impaired insulin secretion, oxidative stress, inflammatory activation, and histopathological disruption. These alterations were associated with suppression of the Nrf2/HO-1/NQO1 antioxidant axis and activation of NF-κB/NLRP3 inflammasome signaling. β-Caryophyllene effectively mitigated these pathological changes in a dose-dependent manner by restoring redox balance, suppressing inflammatory signaling, and preserving pancreatic structural and functional integrity. Collectively, our findings highlight the therapeutic potential of β-caryophyllene as a natural protective agent against mycotoxin-induced pancreatic toxicity and support its relevance in food safety and environmental health contexts. Declarations Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, P.R, A.A.Z and R.S.; methodology, P.R., S.A.A and R.S software, R.S.; validation, P.R., A.A.Z and S.A.A.; formal analysis, A.A.A; investigation, P.R., S.T and S.A.A ; resources, P.R and A.A.Z.; data curation, P.R.; writing—original draft preparation, P.R.; writing—review and editing, A.A.Z and R,S.; visualization, R.S, S.A.A; supervision, P.R.; project administration, P.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement : This study was conducted according to the guidelines of King Faisal University and the “Executive Regulations for Research Ethics on Living Creatures (Second Edition)”, published by the National Bioethics Committee, Saudi Arabia. All animal care and experimental procedures were approved by the Animal Research Ethics Committee at King Faisal University Declaration of King Faisal University and approved by the Institutional Review Board, ETHICS2877. Informed Consent Statement: Not applicable. Data Availability Statement: The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest: The authors declare no conflicts of interest. References Ahmad MS, Alanazi YA, Alrohaimi Y, Shaik RA, Alrashidi S, Al-Ghasham YA, Alkhalifah YS, Ahmad RK (2024) Occurrence, evaluation, and human health risk assessment of ochratoxin A in infant formula and cereal-based baby food: a global literature systematic review. 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Heliyon 9:e19496. https://doi.org/10.1016/j.heliyon.2023.e19496 Sköld M, Karlberg AT, Matura M, Börje A (2006) The fragrance chemical beta-caryophyllene—air oxidation and skin sensitization. Food Chem Toxicol 44:538–545. https://doi.org/10.1016/j.fct.2005.08.028 Son Y, Lee HJ, Ryu D, Kim JR, Kim HY (2024) Ochratoxin A induces hepatic and renal toxicity in mice through increased oxidative stress, mitochondrial damage, and multiple cell death mechanisms. Arch Toxicol 98:2281–2295. https://doi.org/10.1007/s00204-024-03732-3 Wang G, Zhang S, Lan H, Zheng X (2024) Ochratoxin A (OTA) causes intestinal aging damage through the NLRP3 signaling pathway mediated by calcium overload and oxidative stress. Environ Sci Pollut Res Int 31:27864–27882. https://doi.org/10.1007/s11356-024-32696-1 Yang J, Zhou Y, Shi J (2020) Cordycepin protects against acute pancreatitis by modulating NF-κB and NLRP3 inflammasome activation via AMPK. Life Sci 251:117645. https://doi.org/10.1016/j.lfs.2020.117645 Yilmaz N, Verheecke-Vaessen C, Ezekiel CN (2025) Mycotoxins: an ongoing challenge to food safety and security. PLoS Pathog 21:e1013672. https://doi.org/10.1371/journal.ppat.1013672 Yoon JW, Shin S, Park J, Lee BR, Lee SI (2023) TLR/MyD88-mediated inflammation induced in porcine intestinal epithelial cells by ochratoxin A affects intestinal barrier function. Toxics 11:437. https://doi.org/10.3390/toxics11050437 Zanić-Grubisić T, Zrinski R, Cepelak I, Petrik J, Pepeljnjak S (1995) Ochratoxin A impairs activity of the membrane bound enzymes in rat pancreas. Eur J Clin Chem Clin Biochem 33:699–704. https://doi.org/10.1515/cclm.1995.33.10.699 Zhang DD (2025) Thirty years of NRF2: advances and therapeutic challenges. Nat Rev Drug Discov 24:421–444. https://doi.org/10.1038/s41573-025-01145-0 Zhu K, Zhuang Y, Lu H, Song C, Cao X, Cai F, Liu A (2026) Ferroptosis in mycotoxin-induced toxicity: molecular mechanisms, intervention implications, and future directions. J Agric Food Chem 74:1845–1865. https://doi.org/10.1021/acs.jafc.5c08948 Additional Declarations No competing interests reported. Supplementary Files uncroppedWBs.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8885289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596993662,"identity":"c46381d8-484b-4679-9b87-2fb872ccd1f3","order_by":0,"name":"Peramaiyan Rajendran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYJACAwYGZiDFfJihAsQ9QLwWtmSGM8RqYYBo4TEmTou59OEHBR93WOfzz+75bHCwjUGO70YC84cfeLRY9qUZGM48k245487ZzQlALcaSNxLYJHvw+eMMg4Exb9thA4YbuZsPf2xjSNwA1MLAg1cL+wfjv0At8jdyHh8A2lIP1ML88Q9eLTwGxoxALQY3cphBDkswuJHAII3PFssengLD3rZ0A8MbacYGB85JAD32sE1aBo8Wcx72bQY/26wN5G4kP5Y4UGYjz3c8+fDHN/gcBoxCAyS+BBAzNuDRAIn5B3hVjIJRMApGwSgAAGVjUYFQcQJSAAAAAElFTkSuQmCC","orcid":"","institution":"King Faisal University","correspondingAuthor":true,"prefix":"","firstName":"Peramaiyan","middleName":"","lastName":"Rajendran","suffix":""},{"id":596993663,"identity":"b414eac0-0a9f-43df-8f79-a10952c85153","order_by":1,"name":"Abdullah Al Zahrani","email":"","orcid":"","institution":"King Faisal University","correspondingAuthor":false,"prefix":"","firstName":"Abdullah","middleName":"Al","lastName":"Zahrani","suffix":""},{"id":596993674,"identity":"07c72538-53ca-44fe-9484-aafe92a9a9b8","order_by":2,"name":"Ramya Sekar","email":"","orcid":"","institution":"Meenakshi Ammal Dental College \u0026 Hospital, Meenakshi Academy of Higher Education and Research (Deemed to be University)","correspondingAuthor":false,"prefix":"","firstName":"Ramya","middleName":"","lastName":"Sekar","suffix":""},{"id":596993681,"identity":"207979b0-d626-40cf-9fe3-923269dbd727","order_by":3,"name":"Salaheldin Abdelraouf Abdelsalam","email":"","orcid":"","institution":"King Faisal University","correspondingAuthor":false,"prefix":"","firstName":"Salaheldin","middleName":"Abdelraouf","lastName":"Abdelsalam","suffix":""},{"id":596993698,"identity":"e3208c7d-4d06-4230-aadb-4e764e90249e","order_by":4,"name":"Abdulmohsen I. Algefare","email":"","orcid":"","institution":"King Faisal University","correspondingAuthor":false,"prefix":"","firstName":"Abdulmohsen","middleName":"I.","lastName":"Algefare","suffix":""},{"id":596993699,"identity":"c13fba1e-2e71-4758-82db-e555eebf009e","order_by":5,"name":"Sujatha Tejvat","email":"","orcid":"","institution":"King Faisal University","correspondingAuthor":false,"prefix":"","firstName":"Sujatha","middleName":"","lastName":"Tejvat","suffix":""}],"badges":[],"createdAt":"2026-02-15 10:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8885289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8885289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103620439,"identity":"41596b7b-1d5e-4658-87c6-b1e6afef8598","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":874721,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on OTA-Induced Alterations in Glucose Homeostasis. (A) Chemical structure of ochratoxin A (OTA). (B) Chemical structure of β-caryophyllene (BCP). (C) Fasting blood glucose levels. (D) Serum insulin levels. Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p \u0026lt; 0.05 vs. control group; #p \u0026lt; 0.05 vs. OTA group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/127283923d9017005b84ec52.png"},{"id":103620445,"identity":"41f39134-bfbc-493f-8bee-13657e1c4435","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64099,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on OTA-Induced Pancreatic Injury and Systemic Oxidative Stress Markers in Serum. (A) Amylase. (B) Lipase. (C) MDA. (D) TNF-α. Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p \u0026lt; 0.05 vs. control group; #p \u0026lt; 0.05 vs. OTA group.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/4281d8bfeb7874e6ceb0b13b.png"},{"id":103620446,"identity":"d21807ed-dd81-4cd7-8389-a62da580410a","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1202047,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on NLRP3 and NF-κB Signaling Pathways in Pancreatic Tissue. (A) Representative Western blot images showing the expression of NLRP3 and NF-κB proteins. (B) Densitometric analysis of relative protein expression levels of NLRP3 and NF-κB in pancreatic tissue. Protein expression was normalized to β-actin. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p \u0026lt; 0.05 vs. control group; #p \u0026lt; 0.05 vs. OTA group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/4d61efa7dd266fe53e3e0d26.png"},{"id":103620440,"identity":"93a4c729-472c-43a9-9e13-2571401aafb3","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71043,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on Pancreatic Antioxidant Defense in OTA-Exposed Mice. (A) Superoxide dismutase (SOD). (B) Catalase (CAT). (C) Glutathione peroxidase (GPx). (D) Malondialdehyde (MDA). Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p \u0026lt; 0.05 vs. control group; #p \u0026lt; 0.05 vs. OTA group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/29fc5d8844b38af026744fb0.png"},{"id":103620444,"identity":"cc68c5cd-70f3-47d5-a28e-aea9b4f71dec","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8952715,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on Histopathological Alterations in Pancreatic Tissue (H\u0026amp;E Staining). Group I: Control; Group II: OTA; Group III: OTA + BCP (low dose); Group IV: OTA + BCP (high dose); Group V: BCP alone. Histological features include normal serous acini (black arrow), intact fibrovascular septa (yellow arrow), cytoplasmic vacuolation (red arrow), and hemorrhage with vascular congestion (blue arrow). Magnification: 40×; scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/74e7f480219e5d172ca3e4f1.png"},{"id":103620443,"identity":"67b4509d-d37e-4881-bfd7-b53702795d04","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1524727,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BCP on the Nrf2/HO-1/NQO1 Antioxidant Signaling Pathway. (A) Representative Western blot images showing the expression of Nrf2, HO-1, and NQO1 proteins. (B) Densitometric analysis of relative protein expression levels in pancreatic tissue, normalized to β-actin. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p \u0026lt; 0.05 vs. control group; #p \u0026lt; 0.05 vs. OTA group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/6b6344835c82ccdf1f548b25.png"},{"id":105564242,"identity":"c76ee02a-1777-4067-9a02-085903ca02ec","added_by":"auto","created_at":"2026-03-27 12:49:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19138148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/8b414d1b-2e50-4e7d-be20-59b8b13dc6e9.pdf"},{"id":103620442,"identity":"e2250515-9542-4ef6-9eac-b3dd2ce123e9","added_by":"auto","created_at":"2026-02-27 18:07:20","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1395786,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedWBs.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8885289/v1/2a37a7a2706d9a2f9fa8a35f.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-Caryophyllene Protects Against Foodborne Ochratoxin A–Induced Pancreatic Toxicity: Implications for Food Safety and Environmental Health","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe contamination of food and feed by mycotoxins represents a continual global hazard to human and animal health, especially in areas characterized by warm weather and insufficient storage conditions. Among these pollutants, ochratoxin A (OTA) is one of the most widespread and harmful secondary metabolites produced by Aspergillus and Penicillium species. OTA commonly contaminates cereals, coffee, wine, dried fruits, and animal feed, leading to persistent low-dose exposure via the food chain (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Peraica et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). While OTA is primarily known for its nephrotoxic, hepatotoxic, and carcinogenic characteristics, growing evidence suggests that its toxicological effects also impact extra-renal organs, such as the immune system, reproductive tissues, and pancreas (Mor et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2026\u003c/span\u003e; Chen and Wu \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe pancreas is especially vulnerable to xenobiotic-induced damage because of its elevated metabolic requirements, abundant mitochondrial presence, and comparatively restricted antioxidant capability. Experimental studies have shown that OTA alters pancreatic redox equilibrium, causes lipid peroxidation, and inhibits insulin production, ultimately contributing to β-cell dysfunction and metabolic imbalance (Zanić et al. 1995; Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Fakhri et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At the molecular level, exposure to OTA induces excessive formation of reactive oxygen species (ROS), mitochondrial dysfunction, and activation of inflammatory signaling pathways, ultimately resulting in pancreatic tissue damage and cellular apoptosis (Ou et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Son et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The exact processes connecting oxidative stress to the exacerbation of inflammation in OTA-induced pancreatic damage are not fully elucidated. The nuclear factor erythroid 2\u0026ndash;related factor 2 (Nrf2) pathway is a major regulator of cellular antioxidant defense, governing the transcription of numerous cytoprotective genes, including as heme oxygenase-1 (HO-1), superoxide dismutase, catalase, and glutathione-dependent enzymes Kuwar et al. 2025; AlZahrani et al.2024). In toxic or inflammatory conditions, the inhibition of Nrf2 signaling intensifies oxidative damage and enhances tissue susceptibility. In contrast, pharmacological or dietary stimulation of the Nrf2/HO-1 axis has demonstrated considerable protection against chemically induced organ toxicity, including pancreatic damage (Liu et al.2023; Luo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Oxidative stress is a crucial upstream initiator of inflammatory cascades, notably the nuclear factor-κB (NF-κB) pathway and the NOD-like receptor protein 3 (NLRP3) inflammasome, all of which play significant roles in pancreatic inflammation and dysfunction (Yang et el. 2020). Activation of NF-κB enhances the transcription of pro-inflammatory cytokines, whilst the assembly of the NLRP3 inflammasome facilitates caspase-1 activation and the maturation of interleukin-1β and interleukin-18, exacerbating tissue inflammation and damage. Increasing evidence indicates that dysregulated interaction between compromised Nrf2 antioxidant signaling and excessive activation of NF-κB/NLRP3 pathways is crucial in toxin-induced pancreatic injury and metabolic disorders (Papantoniou et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang 2020). Therefore, agents capable of restoring redox balance while simultaneously suppressing inflammatory signaling are of considerable therapeutic interest.\u003c/p\u003e \u003cp\u003eβ-Caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene found in significant quantities in widely used spices, including black pepper, cloves, oregano, and cinnamon (Scandiffio et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sk\u0026ouml;ld et al. 2005). BCP, a bioactive chemical obtained from food, has garnered significant interest due to its antioxidant, anti-inflammatory, and cytoprotective activities, as well as its excellent safety profile (Alonso et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scandiffio et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Preclinical investigations have shown that BCP significantly reduces oxidative stress, inhibits NF-κB activation, suppresses NLRP3 inflammasome signaling, and maintains tissue architecture in diverse models of chemical- and toxin-induced organ injury (Scandiffio et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, recent research suggests that BCP can stimulate the Nrf2/HO-1 pathway, establishing it as a potential dual-action modulator of redox-inflammation imbalance. Although these promising attributes, the preventive function of β-caryophyllene against OTA-induced pancreatic damage has yet to be comprehensively investigated. The potential of BCP to mitigate OTA-induced pancreatic damage via the synchronized activation of Nrf2/HO-1 antioxidant mechanisms and the inhibition of NF-κB/NLRP3 inflammatory signaling is currently uncertain. It is crucial to address this gap to enhance our comprehension of dietary sesquiterpenes as prospective treatments for mycotoxin-related pancreatic and metabolic diseases.\u003c/p\u003e \u003cp\u003eThis study aimed to examine the protective benefits of β-caryophyllene against OTA-induced pancreatic toxicity in a mouse model. We posited that BCP alleviates pancreatic oxidative injury and inflammation via stimulating the Nrf2/HO-1 pathway while suppressing NF-κB/NLRP3 signaling. This study seeks to elucidate the mechanistic understanding of the redox-inflammatory modulation mediated by β-caryophyllene through comprehensive biochemical, molecular, and histopathological analyses, thereby endorsing its prospective use as a dietary or pharmacological intervention for OTA-induced pancreatic damage.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and ethical approval\u003c/h2\u003e \u003cp\u003eAdult male Swiss albino mice (8\u0026ndash;10 weeks old; 25\u0026ndash;30 g) by King Saud University\u0026rsquo;s animal house in Riyadh, Saudi Arabia. These pathogen-free, healthy mice acclimatized for one week in standard conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 60\u0026ndash;70% humidity, 12 h light/dark cycle) within polypropylene cages, with unlimited access to chow and water. All animal experimental procedures were conducted in strict accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and complied with the ARRIVE 2.0 guidelines. The study was conducted in accordance with internationally accepted ethical standards, including the U.K. Animals (Scientific Procedures) Act, 1986 and its associated guidelines, the EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The experimental protocol was reviewed and approved by the Animal Research Ethics Committee of King Faisal University, in accordance with the Declaration of King Faisal University, and approved by the Institutional Review Board (IRB) under approval number: ETHICS2877.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eOchratoxin A (OTA; purity\u0026thinsp;\u0026ge;\u0026thinsp;99%) and β-caryophyllene (BCP; purity\u0026thinsp;\u0026ge;\u0026thinsp;98%) were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Antibodies for Western blotting and immunohistochemistry were sourced from Thermo Fisher Scientific (Waltham, MA, USA). Remaining chemicals and reagents were analytical grade from common commercial sources. OTA was dissolved in [0.5% DMSO in sterile saline] for intraperitoneal administration. BCP was suspended in [olive oil 0.5%] for oral gavage. All other reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design and treatment protocol\u003c/h2\u003e \u003cp\u003eMice were randomly divided into five experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group). Group allocation was coded, and investigators involved in outcome assessment were blinded to treatment.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGroup I (Control): Vehicle 0.5% DMSO (oral gavage)\u003c/p\u003e\u003cp\u003eGroup II (OTA alone): OTA 3mg/kg/day (oral gavage)\u003c/p\u003e\u003cp\u003eGroup III (OTA\u0026thinsp;+\u0026thinsp;BCP low dose): OTA\u0026thinsp;+\u0026thinsp;BCP 25 mg/kg/day (oral gavage)\u003c/p\u003e\u003cp\u003eGroup IV (OTA\u0026thinsp;+\u0026thinsp;BCP-High dose): OTA\u0026thinsp;+\u0026thinsp;BCP high dose 50 mg/kg/day (oral gavage)\u003c/p\u003e\u003cp\u003eGroup V (BCP alone): BCP high dose 50 mg/kg/day (oral gavage)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eOTA was administered orally at [3 mg/kg] [once daily / x 3 day per week] for 6 weeks to induce pancreatic toxicity. BCP was administered by oral gavage daily throughout the OTA exposure period as a co-treatment regimen. The dosing strategy was selected based on published OTA toxicity models and the reported pharmacological safety of BCP (Norah et al. 2024; Hu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Animals were monitored daily for clinical signs of toxicity and mortality. Body weight was recorded weekly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Sample collection\u003c/h2\u003e \u003cp\u003eAt the end of the experimental period, mice were fasted overnight and euthanized under ketamine (80 mg/kg) anesthesia. Blood samples were collected by cardiac puncture, allowed to clot, and centrifuged at 3000 rpm for 10 min at 4\u0026deg;C to obtain serum. The pancreas was rapidly excised, rinsed in ice-cold PBS, blotted dry, and divided into portions for histopathology, biochemical analysis, and protein expression studies. Tissue samples were either fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of pancreatic injury markers\u003c/h2\u003e \u003cp\u003eSerum amylase and lipase levels were measured using commercial diagnostic kits purchased from Cayman Chemical Company (Ann Arbor, MI, USA), according to the manufacturer\u0026rsquo;s instructions. Fasting blood glucose levels were determined using a handheld glucometer, while serum insulin concentrations were quantified using a mouse insulin ELISA kit obtained from Cayman Chemical Company (Ann Arbor, MI, USA) to assess pancreatic endocrine function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Evaluation of oxidative stress and antioxidant enzymes\u003c/h2\u003e \u003cp\u003ePancreatic tissue homogenates (10% w/v) were prepared in ice-cold phosphate buffer (0.1 M, pH 7.4) and centrifuged at 10,000 \u0026times;g for 15 min at 4\u0026deg;C. The supernatant was used to determine MDA levels and the activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) using commercially available assay kits Cayman Chemical Company (Ann Arbor, MI, USA). Protein concentration was measured by the BCA method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Histopathological examination\u003c/h2\u003e \u003cp\u003eFormalin-fixed pancreatic tissues were processed, embedded in paraffin, and sectioned at 4 \u0026micro;m thickness. Sections were stained with hematoxylin and eosin (H\u0026amp;E) and examined under a light microscope Leica DM2500 LED. Pancreatic damage was evaluated by a blinded pathologist using a semi-quantitative scoring system assessing acinar cell degeneration, edema, inflammatory cell infiltration, and vacuolization. Representative photomicrographs were captured at 200\u0026times; magnification and scale bar 50 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blot analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from pancreatic tissue using RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of protein (50 \u0026micro;g) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated overnight at 4\u0026deg;C with primary antibodies against Nrf2, HO-1, NF-κB p65, phospho-NF-κB p65, NLRP3, cleaved caspase-1, IL-1β, IL-18 and β-actin. After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence and quantified by densitometry using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. A \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Analyses were conducted using GraphPad Prism (version v8.4).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. BCP Attenuates OTA-Induced Hyperglycemia in Mice\u003c/h2\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB present the chemical structures of OTA and BCP, respectively. OTA exposure significantly increased fasting blood glucose levels compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating metabolic dysregulation. BCP co-administration attenuated hyperglycemia in a dose-dependent manner, with the high-dose group showing a more pronounced reduction than the low-dose group. Serum insulin analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) revealed a significant decrease in OTA-treated animals (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting impaired β-cell function. BCP treatment significantly restored insulin levels in a dose-dependent fashion, with greater improvement observed at the higher dose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. BCP Mitigates OTA-Induced Pancreatic Injury via Suppression of Oxidative Stress and Inflammation\u003c/h2\u003e \u003cp\u003eSerum amylase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and lipase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) levels were significantly elevated in the OTA-treated group compared with controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), confirming pancreatic exocrine injury. Co-administration of BCP markedly reduced both enzyme levels in a dose-dependent manner, with the high-dose group demonstrating near normalization. OTA exposure also significantly increased serum MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating enhanced lipid peroxidation and oxidative stress (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). BCP treatment significantly attenuated MDA accumulation, suggesting restoration of redox homeostasis, potentially via activation of the Nrf2-mediated antioxidant defense system. Furthermore, serum TNF-α levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) were markedly elevated in OTA-treated animals, reflecting systemic inflammatory activation. BCP supplementation significantly suppressed TNF-α in a dose-dependent manner, supporting inhibition of pro-inflammatory signaling, likely through modulation of the NF-κB pathway. Collectively, these findings indicate that BCP confers pancreatic protection against OTA toxicity by attenuating oxidative stress and inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. BCP Inhibits OTA-Induced Activation of NLRP3 and NF-κB Signaling\u003c/h2\u003e \u003cp\u003eRepresentative Western blot images of NLRP3 and NF-κB proteins are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. OTA exposure markedly upregulated the protein expression levels of both NLRP3 and NF-κB compared with the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating activation of inflammasome formation and pro-inflammatory signaling in pancreatic tissue. Densitometric analysis of band intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), normalized to β-actin, confirmed a significant increase in NLRP3 and NF-κB protein expression in the OTA-treated group. Co-administration of BCP significantly downregulated both proteins in a dose-dependent manner. The high-dose BCP group exhibited a more pronounced suppression compared with the low-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings demonstrate that BCP attenuates OTA-induced pancreatic inflammation by inhibiting NF-κB activation and subsequent NLRP3 inflammasome signaling\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. BCP Restores Pancreatic Antioxidant Defense in OTA-Exposed Mice\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, OTA administration significantly increased pancreatic tissue MDA levels compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating enhanced lipid peroxidation and oxidative damage. Co-treatment with BCP significantly reduced MDA levels in a dose-dependent manner, with the high-dose group exhibiting a more pronounced protective effect. Conversely, antioxidant enzyme activities\u0026mdash;including SOD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), CAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and GPx (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD)\u0026mdash;were significantly decreased in the OTA-treated group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting impaired endogenous antioxidant defense. BCP supplementation markedly restored SOD, CAT, and GPx activities in a dose-dependent fashion, with greater normalization observed at the higher dose. These findings suggest that BCP mitigates OTA-induced oxidative injury by enhancing antioxidant capacity and re-establishing redox balance, likely through activation of the Nrf2-regulated antioxidant signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. BCP on Histopathological Alterations in Pancreatic Tissue\u003c/h2\u003e \u003cp\u003eGroup I (Control): The exocrine pancreas exhibited normal histological architecture, characterized by well-organized serous acini (black arrow) arranged in distinct lobules separated by intact fibrovascular septa (yellow arrow) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Group II (OTA): Pancreatic tissue displayed severe disruption of normal architecture. Marked acinar cell degeneration with prominent cytoplasmic vacuolation (red arrow) was observed. Focal acinar atrophy with areas of necrosis were evident (blue arrow). Additionally, severe interstitial edema accompanied by mononuclear inflammatory cell infiltration and vascular congestion (green arrow) was present. Group III (OTA\u0026thinsp;+\u0026thinsp;BCP Low Dose): Moderate architectural disorganization was noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Acinar cell degeneration with cytoplasmic vacuoles persisted (red arrow). Large focal areas of hemorrhage with vascular congestion were observed (blue arrow), along with mild interstitial infiltration of mononuclear inflammatory cells (green arrow). Group IV (OTA\u0026thinsp;+\u0026thinsp;BCP High Dose): The pancreas showed mild architectural disorganization. Limited areas of acinar degeneration with cytoplasmic vacuolation were present (red arrow), with occasional focal necrosis (green arrow). Mild interstitial fibrosis with sparse mononuclear cell infiltration (blue arrow) was noted, indicating substantial histological improvement compared with the OTA group. Group V (BCP Alone): Pancreatic tissue exhibited normal histological features similar to the control group, with well-preserved serous acini (black arrow) arranged in lobules separated by intact fibrovascular septa (yellow arrow).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. BCP Activates the Nrf2/HO-1/NQO1 Antioxidant Signaling Pathway\u003c/h2\u003e \u003cp\u003eRepresentative Western blot images of Nrf2, HO-1, and NQO1 protein expression are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. OTA exposure significantly reduced the expression levels of Nrf2 and its downstream antioxidant targets, HO-1 and NQO1, compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating suppression of endogenous antioxidant defense mechanisms in pancreatic tissue. Densitometric analysis of band intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), normalized to β-actin, confirmed a significant downregulation of Nrf2, HO-1, and NQO1 in the OTA-treated group. Notably, BCP supplementation markedly restored the expression of these antioxidant proteins in a dose-dependent manner. The high-dose BCP group demonstrated a more pronounced upregulation compared with the low-dose group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggest that BCP confers protection against OTA-induced pancreatic oxidative injury by activating the Nrf2-mediated antioxidant signaling pathway\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMycotoxins are pervasive environmental contaminants produced by toxigenic fungi that proliferate under conditions of high humidity and temperature, making their occurrence particularly relevant in the context of climate change and global food insecurity (Marroqu\u0026iacute;n et al 2014; Casu et al \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shetty et al \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Environmental factors such as improper storage, agricultural practices, and rising temperatures significantly influence fungal growth and toxin production, leading to contamination of cereals, nuts, and animal feed. Chronic exposure to mycotoxins through the food chain poses substantial public health risks, including hepatotoxicity, nephrotoxicity, immunosuppression, and metabolic dysfunction (Awuchi et al \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yilmaz et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, understanding the environmental determinants of mycotoxin contamination and developing effective mitigation strategies are critical for safeguarding food safety and reducing toxin-induced organ damage.\u003c/p\u003e \u003cp\u003eThe present study demonstrates that BCP effectively mitigates OTA-induced pancreatic toxicity through coordinated modulation of oxidative stress, inflammatory signaling, and inflammasome activation. OTA exposure resulted in marked hyperglycemia, reduced serum insulin levels, elevated pancreatic injury markers (amylase and lipase), enhanced lipid peroxidation, and suppression of endogenous antioxidant defenses. These biochemical alterations were accompanied by severe histopathological damage and activation of NF-κB/NLRP3 signaling, collectively indicating pancreatic dysfunction driven by oxidative and inflammatory mechanisms. OTA is a well-recognized mycotoxin that induces organ toxicity primarily via excessive ROS generation and redox imbalance. In the present study, elevated MDA levels together with decreased SOD, CAT, and GPx activities confirm oxidative stress as a central mechanism of OTA-induced pancreatic injury. The suppression of Nrf2 and its downstream targets HO-1 and NQO1 further supports impairment of the intrinsic antioxidant defense system. Nrf2 is a master regulator of cellular redox homeostasis, and its downregulation enhances susceptibility to oxidative damage and metabolic dysregulation (Goel et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al 2025).\u003c/p\u003e \u003cp\u003eInflammation also played a critical role in OTA-mediated toxicity. Increased TNF-α levels and upregulated NF-κB expression indicate activation of pro-inflammatory transcriptional pathways. NF-κB is a pivotal regulator of cytokine production and inflammasome priming (Yoon et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consistently, the elevated expression of NLRP3 suggests activation of the inflammasome complex, which amplifies inflammatory cascades and contributes to tissue degeneration (Wang et al 2025). Histopathological findings including acinar degeneration, necrosis, edema, and inflammatory infiltration corroborate the biochemical and molecular results. Importantly, BCP supplementation significantly attenuated OTA-induced pancreatic injury in a dose-dependent manner. BCP reduced hyperglycemia and restored serum insulin levels, suggesting preservation of endocrine function. It also normalized amylase and lipase levels, indicating protection of exocrine integrity. At the molecular level, BCP suppressed NF-κB activation and downregulated NLRP3 expression, thereby limiting inflammatory amplification. Concurrently, BCP restored Nrf2, HO-1, and NQO1 expression, enhancing antioxidant capacity and re-establishing redox balance. The reciprocal regulation between Nrf2 and NF-κB pathways likely underlies the protective mechanism of BCP. Activation of Nrf2 not only enhances antioxidant defense but also indirectly suppresses NF-κB mediated inflammatory signaling. Therefore, BCP appears to exert dual protective actions by both scavenging oxidative stress and inhibiting inflammatory signaling cascades. These findings are consistent with previous reports describing BCP as a natural sesquiterpene with potent antioxidant and anti-inflammatory properties.\u003c/p\u003e \u003cp\u003eCollectively, the present data indicate that OTA-induced pancreatic toxicity is mediated by oxidative stress driven NF-κB activation and subsequent NLRP3 inflammasome signaling, whereas BCP confers significant protection through activation of the Nrf2/HO-1/NQO1 axis and suppression of inflammatory pathways. These findings highlight the therapeutic potential of BCP as a natural protective agent against mycotoxin-induced metabolic and pancreatic dysfunction.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, the present study demonstrates that ochratoxin A induces significant pancreatic injury characterized by hyperglycemia, impaired insulin secretion, oxidative stress, inflammatory activation, and histopathological disruption. These alterations were associated with suppression of the Nrf2/HO-1/NQO1 antioxidant axis and activation of NF-κB/NLRP3 inflammasome signaling. β-Caryophyllene effectively mitigated these pathological changes in a dose-dependent manner by restoring redox balance, suppressing inflammatory signaling, and preserving pancreatic structural and functional integrity. Collectively, our findings highlight the therapeutic potential of β-caryophyllene as a natural protective agent against mycotoxin-induced pancreatic toxicity and support its relevance in food safety and environmental health contexts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used \u0026ldquo;Conceptualization, P.R, A.A.Z and R.S.; \u0026nbsp; \u0026nbsp;methodology, P.R., S.A.A and R.S software, R.S.; validation, P.R., A.A.Z and S.A.A.; formal analysis, A.A.A; investigation, P.R., S.T and S.A.A ; resources, P.R and A.A.Z.; data curation, P.R.; writing\u0026mdash;original draft preparation, P.R.; writing\u0026mdash;review and editing, A.A.Z and R,S.; visualization, R.S, S.A.A; supervision, P.R.; project administration, P.R. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e This study was conducted according to the guidelines of King Faisal University and the \u0026ldquo;Executive Regulations for Research Ethics on Living Creatures (Second Edition)\u0026rdquo;, published by the National Bioethics Committee, Saudi Arabia. All animal care and experimental procedures were approved by the Animal Research Ethics Committee at King Faisal University Declaration of King Faisal University and approved by the Institutional Review Board, ETHICS2877.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmad MS, Alanazi YA, Alrohaimi Y, Shaik RA, Alrashidi S, Al-Ghasham YA, Alkhalifah YS, Ahmad RK (2024) Occurrence, evaluation, and human health risk assessment of ochratoxin A in infant formula and cereal-based baby food: a global literature systematic review. 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J Agric Food Chem 74:1845\u0026ndash;1865. https://doi.org/10.1021/acs.jafc.5c08948\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mycotoxin, Ochratoxin A, β-Caryophyllene, Pancreas, NF-κB/NLRP3","lastPublishedDoi":"10.21203/rs.3.rs-8885289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8885289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing prevalence of mycotoxin contamination due to climate change and environmental stressors represents a growing global health concern, necessitating effective preventive and therapeutic strategies. Ochratoxin A (OTA) is a pervasive foodborne mycotoxin linked to multisystem toxicity; nonetheless, its harmful effects on pancreatic tissue and the underlying molecular processes are inadequately defined. Increasing evidence indicates that oxidative stress and sterile inflammation produced by OTA are pivotal in pancreatic damage. β-Caryophyllene (BCP), a natural sesquiterpene found in consumable flora, demonstrates significant antioxidant and anti-inflammatory effects. The objective of this study was to examine the protective effect of BCP against OTA-induced pancreatic toxicity, focusing on the modulation of the Nrf2/HO-1 antioxidant pathway and the NF-κB-mediated activation of the NLRP3 inflammasome. Experimental animals were subjected to OTA to elicit pancreatic toxicity and simultaneously administered BCP at both low and high dosages. Pancreatic damage was assessed via serum biochemical markers and histological analysis. Parameters of oxidative stress and inflammatory mediators were measured in pancreatic tissue. Protein expression levels of Nrf2, HO-1, NF-κB, and NLRP3 signaling components were examined to clarify the molecular foundation of BCP-induced protection. OTA exposure caused significant pancreatic damage, indicated by increased oxidative stress, activation of NF-κB and NLRP3 inflammasome signaling, and notable histoarchitectural disruption. BCP therapy markedly reduced OTA-induced pancreatic damage in a dose-dependent fashion. BCP augmented intrinsic antioxidant defenses through the activation of the Nrf2/HO-1 pathway while simultaneously inhibiting NF-κB activation and subsequent NLRP3 inflammasome signaling. BCP significantly alleviates OTA-induced pancreatic damage by reestablishing redox equilibrium and suppressing inflammatory signaling pathways. These findings offer new understanding of OTA-induced pancreatic damage and recognize BCP as a potential dietary strategy for mitigating mycotoxin-related pancreatic impairment, with possible implications for health hazards connected with environmental and food safety.\u003c/p\u003e","manuscriptTitle":"β-Caryophyllene Protects Against Foodborne Ochratoxin A–Induced Pancreatic Toxicity: Implications for Food Safety and Environmental Health","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 18:07:15","doi":"10.21203/rs.3.rs-8885289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f35c5f9-3760-4ccb-9280-8488349e6983","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T08:58:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 18:07:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8885289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8885289","identity":"rs-8885289","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}